The Homeobox transcription factor HLX1 is essential for hematopoietic development

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Short Talk Presentations/ Experimental Hematology 44 (2016) S40–S54

2005 - ROLE OF ENDOGENOUS HISTONE METHYLTRANSFERASES IN LEUKEMIA Patricia Ernst1, Yufei Chen2, Konstantinos Anastassiadis3, Andrea Krantz3, Akihiko Yokoyama4, A. Francis Stewart3, Tobias Neff1, and Kenneth Jones1 1 University of Colorado, Denver, Denver USA; 2Dartmouth and University of Colorado, Denver, Denver, USA; 3Technische Universit€at Dresden, Dresden, Germany; 4Kyoto University School of Medicine Medical Innovation Center, Kyoto, Japan MLL1 chromosomal translocations occur frequently in infant leukemia and in adult therapy-induced leukemia. Due to the poor prognosis of MLL-rearranged (MLLr) leukemia, significant effort has gone into developing targeted therapeutics to inhibit MLL-fusion proteins (FPs), which are expressed from the most common MLL1 rearrangements. The remaining wild-type MLL1 allele is typically preserved and expressed in MLL-FP leukemia blasts, suggesting functional importance of the wildtype MLL1 protein. In addition, published studies suggest that the recruitment or activity of MLL-FPs may depend on the activity of the remaining MLL1 allele. Our recent studies demonstrate that MLL-AF9-driven leukemogenesis progresses normally in cells genetically engineered to lack the HMT domain of MLL1. These and other observations call into question the role of MLL1 in MLL-FP-initiated leukemia, prompting us to carefully revisit the role of wild-type MLL1 in MLL-FP-initiated leukemia using genetic models. To directly address whether endogenous MLL1 plays any role in facilitating transformation by MLL-FPs, we employed conditional mutagenesis to delete Mll1 or a critical cofactor, Menin (Men1), in the context of ongoing acute myelogenous leukemia (AML) initiated by either MLL-AF9 or MLL-AF6. Men1 encodes a protein that directs chromatin targeting of both wild-type MLL1 and MLL-FP complexes. Surprisingly, we find that Mll1 is dispensable for MLL-AF9- and MLL-AF6-mediated AML progression in vitro or in vivo. In contrast, deletion of Men1 significantly impairs leukemia proliferation and survival, both in vivo and in vitro, as others have observed. Furthermore, the lack of requirement for endogenous Mll1 was confirmed using two distinct Mll1 conditional knockout alleles. Upon Mll1 deletion few gene expression changes occurred, and MLL-AF9 fusion targets were absolutely unaffected by Mll1 deletion. This result contrasted with striking gene expression changes, cell cycle arrest and ultimately death of AML blasts that occurred upon deletion of Men1. This surprising lack of impact of Mll1 deletion prompted us to consider redundancy with the related methyltransferase Mll2. First, loss of Mll2 alone affected leukemia survival, but co-deletion very effectively killed leukemia cells. Unexpectedly, this was not due to redundancy between the Mll genes as genome wide data indicated they regulate largely distinct sets of genes, which in neither case included MLLAF9 direct targets. Furthermore, MLL2, but not MLL1 loss led to a global reduction in H3K4me3/me2 in MLL-AF9 leukemia cells. Overall, our data demonstrate that endogenous MLL1 is not required for maintaining leukemogenesis in MLL-FP-initiated leukemia. These data argue against the utility of drugs that exclusively target the wild-type MLL1 protein complex in the treatment of MLLr leukemia, whereas inhibition of both MLL1 and MLL2 methyltransferases may significantly impair leukemia survival.

2007 - THERAPEUTIC BLOCKADE OF MACROPHAGE COLONY STIMULATING FACTOR (CSF-1) DELAYS LEUKAEMIA PROGRESSION OF AML IN MICE IN VIVO Sal Lee Goh1, Jean- Pierre Levesque1, Allison Pettit1, Valerie Barbier1, Cecile Jeanclos2, and Ingrid Winkler1 1 MRI-UQ, Australia; 2MMRI, Sunnyvale, USA Macrophage colony-stimulating factor (M-CSF or CSF-1) plays a role in regulating innate immune responses promoting macrophage growth and differentiation. We hypothesized CSF-1 may also play a role in growth and progression of Acute Myeloid Leukaemia (AML). The aim of this study is to investigate the role of CSF-1 in survival and chemo-resistance of leukaemia stem and progenitor cells (LSPC). We further hypothesized that blocking CSF-1R signalling in LSPC may dampen leukaemia survival in vitro and delay leukaemia progression in vivo. AML was induced in mice by injecting murine Haematopoietic Stem and Progenitor Cells (HSPC) transduced with either MLL-AF9 or AML1- ETO fusion oncogenes for the development of either monomyelocytic or granulocytic leukaemia respectively. We found CSF-1R, the main receptor for CSF-1, was expressed on these acute myeloid leukaemia cells, thus it is possible that CSF-1 provide supportive microenvironment for leukemic growth. To identify whether CSF-1 in the bone marrow (BM) niche is essential for growth of malignant LSPC, we harvested normal or leukaemic blasts from BM for in vitro studies using Long-Term Culture-Initiating-Cell (LT-CIC) assays. In these assays AML LSPC or normal HSPC cells were co-cultured with mesenchymal stromal cells (MSC) from either wildtype mice (MSC that produce CSF-1) or MSC from OP/OP mice (unable to produce functional CSF-1). We found normal (wild-type) HSPC were able to proliferate, survive and produce long-term colony initiating cells (LT-CIC) in the absence of niche-provided CSF-1, however AML blasts could not, unless rescued by addition of recombinant CSF-1 (100 ng/mL) in vitro. Together these data suggest CSF-1 signalling may be critical for AML LSPC but not normal HSPC. Next we investigated in mice whether therapeutic CSF-1 blockade could similarly dampen AML survival or progression in vivo. Cohorts of mice were injected with luciferase-expressing monomyelocytic (MLL-AF9) BM leukaemic blasts, then 7 days later administered the small molecule CSF-1 antagonist (GW2580, 160mg/kg daily for 10 days) or vehicle control. Leukaemia progression was tracked by biweekly bioluminescence and testbleeds for appearance of GFP+ leukaemia blasts in blood. We found therapeutic blockade of CSF-1 significantly reduced tumour burden in these mice by both bioluminesce and testbleed analysis. Mice were also monitored for duration of survival. As anticipated by the observed reduction in leukaemia burden, therapeutic CSF-1 blockade also significantly extended the duration of overall mouse survival (P ! 0.005, n5 8 mice/ group). Together these studies suggest therapeutic CSF-1 blockade may show promise as an adjunct therapy to help reduce tumour burden and improve success of AML leukaemia therapies.

2008 - THE HOMEOBOX TRANSCRIPTION FACTOR HLX1 IS ESSENTIAL FOR HEMATOPOIETIC DEVELOPMENT Eirini Trompouki1, Indre Piragyte1, Alexandros Polyzos2, and Na Yin1 1 Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; 2Biomedical Research Foundation, Academy of Athens, Athens, Greece Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-Like Homeobox) is frequently observed in patients with acute myeloid leukemia. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a critical role during hematopoietic formation and differentiation in zebrafish. Zebrafish hlx1 morphants have normal primitive hematopoietic specification, but show a decline in erythrocytes and concomitant increase in myeloid cells at 48 hpf, suggesting a crucial role of hlx1 during the formation of intermediate erythromyeloid precursors (EMPs). Since EMPs arise from endothelial precursor cells, where hlx1 is primarily expressed, we performed expression analysis of sorted endothelial cells. Interestingly, we observed that many genes involved in erythropoiesis are expressed in wild-type endothelial cells but the whole erythroid program and heme biosynthesis related genes are massively downregulated in hlx1 morphants. Chromatin immunopreciptation assays in human cell lines show that HLX binds to putative enhancers and regulatory elements of erythropoietic regulators like TAL1, establishing HLX as a direct regulator of the erythroid program. hlx1 morphants have also diminished numbers of definitive hematopoietic stem and progenitor cells (HSPCs), resulting in fewer thymocytes. Overexpression of HLX in endothelial cells leads to rescue of the morphant phenotype and results in aberrant production of HSPCs. Surprisingly, hlx1 morphants exhibit diminished ribosomal protein production and attenuation of

Short Talk Presentations/ Experimental Hematology 44 (2016) S40–S54

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metabolic and unfolded protein response pathways that account, at least partially, for the HSPCs phenotype. Together, our data suggest that hlx1 controls the erythromyeloid decision during EMP formation and positively regulates HSPCs formation by fine-tuning metabolic pathways.

macrophages are severely depleted in all tissues analyzed. In the dorsal aorta, Sl/Sl embryos have smaller and less proliferative hematopoietic clusters and display a delay in maturation of phenotypic Pre-HSC I into Pre-HSC II. Thus, our in vivo data identify a previously unrecognized role for KitL prior to HSC-derived hematopoiesis in the fetal liver. Since EMPs sustain erythropoiesis in mid-gestation murine fetuses, this implies that the previously described anaemic phenotype observed in the fetal liver of Sl/Sl mutants is EMP-derived, rather than HSC-derived. Our ongoing studies aim to further elucidate the mechanism by which KitL exerts its role in early hematopoiesis, and to identify other candidate cytokines with possible synergistic effects.

2009 - EMBRYONIC HEMATOPOIETIC PROGENITORS DEPEND ON KIT LIGAND FOR IN VIVO EXPANSION AND DIFFERENTIATION Emanuele Azzoni1, Joe Harman2, Joana Carrelha2, Kathleen McGrath3, Claus Nerlov2, James Palis4, Sten Eirik Jacobsen5, and Marella De Bruijn2 1 MRC Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK; 2University of Oxford, Oxford, UK; 3University of Rochester Medical Center, Rochester, NY, USA; 4University of Rochester Medical Center, Rochester, USA; 5 University of Oxford, Karolinska Institutet, Oxford, UK

2010 - RADIO-RESISTANT RECIPIENT BONE MARROW (BM) MACROPHAGES (MACS) ARE NECESSARY FOR HEMATOPOIETIC STEM CELL (HSC) ENGRAFTMENT POST TRANSPLANTATION Jean-Pierre Levesque1, Simranpreet Kaur1, Rebecca Jacobsen1, Susan Millard1, Lena Batoon1, Ingrid Winkler1, Kelli Macdonald2, Andrew Perkins1, David Hume3, Liza Raggatt1, and Allison Pettit1 1 Mater Research Institute - University of Queensland, Australia; 2 Queensland Institute of Medical Research, Brisbane, Australia; 3Roslin Institute - The University of Edinburgh, Edinburgh, UK

Embryonic hematopoiesis in mammals takes place in multiple anatomical sites, through distinct but overlapping waves. During this process, a variety of hematopoietic progenitors are generated, including erythro-myeloid progenitors (EMPs) and hematopoietic stem cells (HSCs). The micro-environmental and extrinsic factors that orchestrate the establishment of a functional hematopoietic system are still poorly understood. Kit ligand (KitL; also known as Stem Cell Factor/SCF) is a cytokine that plays a pivotal role in the maintenance of adult hematopoiesis. Furthermore, the absence of KitL in utero causes a significant reduction in the HSC pool and embryos die with severe anaemia. However, it is currently unclear how and when hematopoietic defects originate in embryos lacking KitL, and what is the precise role of this cytokine in the first steps of hematopoiesis. Using a novel KitL transgenic reporter line, we show that KitL-expressing cells are found in the microenvironment of all hematopoietic sites of the E8.5-E10.5 mouse embryo. We next investigated in detail the hematopoietic phenotype of early Steel (Sl/Sl) mutant embryos, which lack KitL. In these embryos, anemia is not detectable until E13.5 or later, meaning that the primitive wave of erythropoiesis does not require Kit signalling. We show that in Sl/Sl embryos the emergence of EMPs in the yolk sac is unaffected, but their subsequent expansion and differentiation in the fetal liver are severely compromised. These embryos display a dramatic reduction in fetal liver erythroid cells prior to the onset of HSC-derived erythropoiesis. Imaging flow cytometry analysis revealed a severe loss of CD71+ Kit+ Ter119lo early progenitors, but normal proportions of late stage erythroblasts within the reduced total number of erythroid cells, indicating that the bottleneck is at the early progenitor level. Strikingly, EMP-derived tissue resident

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BMMacs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-LinKit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using